Efficient production of heterologous proteins using mannosyl transferase inhibitors

ABSTRACT

Compounds and methods are described for producing protein compositions having reduced amounts of O-linked glycosylation. The method includes producing the protein in cells cultured in the presence of certain benzylidene thiazolidinediones inhibitors of Pmt-mediated O-linked glycosylation.

BACKGROUND OF THE INVENTION

Glycoproteins mediate many essential functions in humans and othermammals, including catalysis, signaling, cell-cell communication, andmolecular recognition and association. Glycoproteins make up themajority of non-cytosolic proteins in eukaryotic organisms (Lis Sharon,1993, Eur. J. Biochem. 218:1-27). Many glycoproteins have been exploitedfor therapeutic purposes, and during the last two decades, recombinantversions of naturally-occurring glycoproteins have been a major part ofthe biotechnology industry. Examples of recombinant glycosylatedproteins used as therapeutics include erythropoietin (EPO), therapeuticmonoclonal antibodies (mAbs), tissue plasminogen activator (tPA),interferon-β (IFN-β), granulocyte-macrophage colony stimulating factor(GM-CSF), and human chorionic gonadotrophin (hCH) (Cumming et al., 1991,Glycobiology 1:115-130). Variations in glycosylation patterns ofrecombinantly produced glycoproteins have recently been the topic ofmuch attention in the scientific community as recombinant proteinsproduced as potential prophylactics and therapeutics approach theclinic.

In general, the glycosylation structures of glycoproteinoligosaccharides will vary depending upon the host species of the cellsused to produce them. Therapeutic proteins produced in non-human hostcells are likely to contain non-human glycosylation which may elicit animmunogenic response in humans—e.g. hypermannosylation in yeast (Ballou,1990, Methods Enzymol. 185:440-470); α(1,3)-fucose and β(1,2)-xylose inplants, (Cabanes-Macheteau et al., 1999. Glycobiology, 9: 365-372);N-glycolylneuraminic acid in Chinese hamster ovary cells (Noguchi etal., 1995. J. Biochem. 117: 5-62); and, Galα-1,3Gal glycosylation inmice (Borrebaeck, et ad., 1993, Immun. Today, 14: 477-479). Carbohydratechains bound to proteins in animal cells include N-glycoside bond typecarbohydrate chains (also called N-glycans; or N-linked glycosylation)bound to an asparagine (Asn) residue in the protein and O-glycoside bondtype carbohydrate chains (also called O-glycans; or O-linkedglycosylation) bound to a serine (Ser) or threonine (Thr) residue in theprotein.

Because the oligosaccharide structures of glycoproteins produced bynon-human mammalian cells tend to be more closely related to those ofhuman glycoproteins, most commercial glycoproteins are produced inmammalian cells. However, mammalian cells have several importantdisadvantages as host cells for protein production. Besides beingcostly, processes for producing proteins in mammalian cells produceheterogeneous populations of glycoforms, have low volumetric titers, andrequire both ongoing viral containment and significant time to generatestable cell lines.

It is well recognized that the particular glycoforms on a protein canprofoundly affect the properties of the protein, including itspharmacokinetic, pharmacodynamic, receptor-interaction, andtissue-specific targeting properties (Graddis et al., 2002. Curr PharmBiotechnol. 3: 285-297). For example, it has been shown that differentglycosylation patterns of Igs are associated with different biologicalproperties (Jefferis and Lund, 1997, Antibody Eng. Chem. Immunol., 65;111-128; Wright and Morrison, 1997, Trends Biotechnol., 15: 26-32). Ithas further been shown that galactosylation of a glycoprotein can varywith cell culture conditions, which may render some glycoproteincompositions immunogenic depending on the specific galactose pattern onthe glycoprotein (Patel et al., 1992. Biochem J. 285: 839-845). However,because it is not known which specific glycoform(s) contribute(s) to adesired biological function, the ability to enrich for specificglycoforms on glycoproteins is highly desirable. Because differentglycoforms are associated with different biological properties, theability to enrich for glycoproteins having a specific glycoform can beused to elucidate the relationship between a specific glycoform and aspecific biological function of the glycoprotein. Also, the ability toenrich for glycoproteins having a specific glycoform enables theproduction of therapeutic glycoproteins having particular specificities.Thus, production of glycoprotein compositions that are enriched forparticular glycoforms is highly desirable.

While the pathway for N-linked glycosylation has been the subject ofmuch analysis, the process and function of O-linked glycosylation is notas well understood. However, it is known that in contrast to N-linkedglycosylation, β-glycosylation is a posttranslational event, whichoccurs in the cis-Golgi (Varki, 1993, Glycobiol., 3: 97-130). While aconsensus acceptor sequence for O-linked glycosylation like that forN-linked glycosylation does not appear to exist, a comparison of aminoacid sequences around a large number of O-linked glycosylation sites ofseveral glycoproteins show an increased frequency of proline residues atpositions −1 and +3 relative to the glycosylated residues and a markedincrease of serine, threonine, and alanine residues (Wilson et al.,1991, Biochem. J., 275: 529-534). Stretches of serine and threonineresidues in glycoproteins, may also be potential sites forO-glycosylation.

One gene family that has a role in O-linked glycosylation are the genesencoding the Dol-P-Man:Protein (Ser/Thr) Mannosyl Transferase (Pmt).These highly conserved genes have been identified in both highereukaryotes such as humans, rodents, insects, and the like and lowereukaryotes such as fungi and the like. Yeast such as Saccharomycescerevisiae and Pichia pastoris encode up to seven PMT genes encoding Pmthomologues (reviewed in Willer et al. Curr. Opin. Struct. Biol. 2003October; 13(5): 621-30.). In yeast, O-linked glycosylation starts by theaddition of the initial mannose from dolichol-phosphate mannose to aserine or threonine residue of a nascent glycoprotein in the endoplasmicreticulum by one of the seven O-mannosyl transferases genes. While thereappear to be seven PMT genes encoding Pmt homologues in yeast,O-mannosylation of secreted fungal and heterologous proteins in yeast isprimarily dependent on the genes encoding Pmt1 and Pmt2, which appear tofunction as a heterodimer. PMT1 and PMT2 and their protein products,Pmt1 and Pmt2, respectively, appear to be highly conserved amongspecies.

Tanner et al. in U.S. Pat. No. 5,714,377 describes the PMT1 and PMT2genes of Saccharomyces cerevisiae and a method for making recombinantproteins having reduced O-linked glycosylation that uses fungal cells inwhich one or more of PMT genes have been genetically modified so thatrecombinant proteins are produced, which have reduced O-linkedglycosylation.

Ng et al. in U.S. Published Patent Application No. 20020068325 disclosesinhibition of O-glycosylation through the use of antisense orcosuppression or through the engineering of yeast host strains that haveloss of function mutations in genes associated with O-linkedglycosylation, in particular, one or more of the PMT genes.

UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyl-transferases (GalNAc-transferases) are involved in mucintype O-linked glycosylation found in higher eukaryotes. These enzymesinitiate O-glycosylation of specific serine and threonine amino acids inproteins by adding N-acetylgalactosamine to the hydroxy group of theseamino acids to which mannose residues can then be added in a step-wisemanner. Clausen et al. in U.S. Pat. No. 5,871,990 and U.S. PublishedPatent Application No. 20050026266 discloses a family of nucleic acidsencoding UDP-N-acetyl-alpha-D-galactosamine:polypeptide N-acetylgalactosaminyl-transferases (GalNAc-transferases). Clausen in U.S.Published Patent Application No. 20030186850 discloses the use ofGalNAc-beta-benzyl to selectively inhibit lectins of polypeptideGalNAc-transferases and not serve as substrates for otherglycosyltransferases involved in O-glycan biosyntheses, thus inhibitingO-glycosylation.

Inhibitors of O-linked glycosylation have been described. For example,Orchard et al. in U.S. Pat. No. 7,105,554 describes benzylidenethiazolidinediones and their use as antimycotic agents, e.g., antifungalagents. These benzylidene thiazolidinediones are reported to inhibit thePmt1 enzyme, preventing the formation of the O-linked mannoproteins andcompromising the integrity of the fungal cell wall. The end result iscell swelling and ultimately death through rupture.

Konrad et al. in U.S. Published Patent Application No. 20020128235disclose a method for treating or preventing diabetes mellitus bypharmacologically inhibiting O-linked protein glycosylation in a tissueor cell. The method relies on treating a diabetic individual with(Z)-1-[N-(3-Ammoniopropyl)-N-(n-propyl)amino]diazen-ium-1,2-diolate or aderivative thereof, which binds O-linked N-acetylglucosamine transferaseand thereby inhibits O-linked glycosylation.

Kojima et al. in U.S. Pat. No. 5,268,364 disclose therapeuticcompositions for inhibition of O-glycosylation using compounds such asbenzyle-α-N-acetylgalactosamine, which inhibits extension ofO-glycosylation leading to accumulation of O-α-GalNAc, to blockexpression of SLex or SLea by leukocytes or tumor cells and therebyinhibit adhesion of these cells to endothelial cells and platelets.

Boime et al. U.S. Pat. No. 6,103,501 disclose variants of hormones inwhich O-linked glycosylation was altered by modifying the amino acidsequence at the site of glycosylation.

The invention is directed to novel inhibitors of Pmt proteins, which areuseful for production of recombinant proteins with reduced O-linkedglycosylation. This enables O-linked glycosylation of proteins producedfrom fungi and yeast cells to be controlled.

SUMMARY OF THE INVENTION

Compounds and methods are described for producing protein compositionshaving reduced amounts of O-linked glycosylation. The method includesproducing the protein in cells cultured in the presence of certainbenzylidene thiazolidinediones inhibitors of Pmt-mediated O-linkedglycosylation.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 illustrates the effect of Pmt inhibitors including the novelinhibitor shown as Example 4 on O-glycosylation of secreted recombinantmonoclonal antibody in Pichia pastoris. The chemical inhibitors of Pmtreduced O-glycosylation in a dose-dependent fashion. Western blottingusing an anti-human H+L antibody was used to detect heavy (Hc) and light(Lc) chains in the growth media of strains treated with increasingamounts of Pmt inhibitor (higher doses in left lanes). The slowestmigrating band (marked with asterisk) indicates the O-glycosylated Hcwhich is eliminated by Pmt inhibitor treatment.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides novel compounds and methods forexpressing a recombinant protein (includes polypeptides andglycoproteins), which is susceptible to O-linked glycosylation in aparticular host cell, having a reduced amount of O-linked glycosylation(including no O-linked glycosylation) in that cell type. The methodinvolves inducing expression of a protein of interest in a host cell inwhich the protein is susceptible to O-linked glycosylation in the hostcell in the presence of one or more novel compounds of the inventionwhich are inhibitors of the activity of one or more of theDol-P-Man:Protein (Ser/Thr) Mannosyl Transferase (Pmt) proteins involvedin the transfer of mannose to a serine or threonine residue of theprotein in the cell, optionally with one or more a 1,2-mannosidases asdescribed in Bobrowicz et al. U.S. Published Application No. 2007061631,at the time expression of the protein is induced. The protein that isexpressed in the presence of the inhibitor has a reduced amount ofO-linked glycosylation compared to the amount of O-linked glycosylationthat would have been present on the protein if it had been produced inthe absence of the inhibitor. The method is particularly useful becauseit provides a means for producing therapeutically relevant proteinswhere it is desired that the protein have a reduced amount ofO-glycosylation in host cells such as lower eukaryotes, for exampleyeast, and bacteria, which would normally produce proteins with O-linkedglycans, having a reduced number of O-linked glycans. However, while themethod is especially suitable for expressing proteins with reducedO-linked glycosylation in lower eukaryotic organisms, the method canalso be practiced in higher eukaryotic organisms and bacteria.

The Pmt inhibitors of the invention are selected from the followinggroup:

or a salt thereof.

The methods described herein are an improvement over certain prior artmethods for producing proteins having reduced O-linked glycosylation inhost cells in which the proteins are susceptible to O-linkedglycosylation. For example, Tanner et al. in U.S. Pat. No. 5,714,377describes a method for making recombinant proteins having reducedO-linked glycosylation using fungal cells such as yeast cells in whichone or more of PMT genes encoding the Pmt protein have been geneticallymodified so that recombinant proteins are produced which have reducedO-linked glycosylation. While deletion of the PMT1, PMT2, or PMT4 genesin a fungal host cell enables production of a recombinant protein havingreduced O-linked glycosylation in the fungal host cell, expression ofthe PMT genes are important for host cell growth and either deletionalone also adversely affects the ability of the fungal host cell to growthus making it difficult to produce a sufficient quantity of host cellsor recombinant protein with a reduced amount of O-linked glycosylation.Deletion of PMT2 plus either PMT1 or PMT4 appears to be lethal to thefungal host cell. Therefore, genetic elimination of the PMT genes in ahost cell would appear to be an undesirable means for producingrecombinant proteins having reduced O-linked glycosylation.

In contrast, the PMT genes in the host cells used in the methods of thepresent invention have not been modified or deleted, which enables thehost cell to O-glycosylate those proteins that are important for cellgrowth until which time the activity of the Pmt proteins is inhibited.In general, this enables the host cells to be grown to higher levelsthan the levels that could be obtained if the PMT genes had beendeleted. In addition, in particular embodiments, expression of therecombinant protein in the host cell is controlled by an induciblepromoter and the Pmt activity in the host cell is not inhibited untilexpression of the recombinant protein is induced. This enables largequantities of host cells containing a nucleic acid encoding arecombinant protein to be produced in culture before inducing expressionof the recombinant protein and adding the Pmt inhibitor. This can enableproduction of larger amounts recombinant protein having reduced O-linkedglycosylation to be produced in the culture in a shorter period of timethan would occur for host cells which have had one or more PMT genesdeleted and grow poorly in culture.

The Pmt inhibitors described herein are improved over those describedpreviously in Orchard et al. (U.S. Pat. No. 7,105,554) and Bobrowicz etal (U.S. Published Application No. 2007061631) based on increasedpotency as shown in Example 5. The increased potency allows for the useof smaller amounts of inhibitor to reduce fungal O-glycosylation toacceptable levels and/or increases the potential for completeelimination of O-glycans.

The methods described herein facilitate the production of glycoproteinshaving reduced O-linked glycosylation in host cells that have beengenetically modified to produce glycoproteins having predominantly aparticular N-linked glycan structure but which also O-glycosylate theglycoprotein. Methods for producing a wide variety of glycoproteinshaving predominantly particular N-linked glycoforms have been disclosedin U.S. Pat. No. 7,029,872 and U.S. Published Application Nos.20050170452, 20050260729, 20040230042, 20050208617, 20050208617,20040171826, 20060160179, 20060040353, and 20060211085. Any one of thehost cells described in the aforementioned patent and patentapplications can be used to produce a glycoprotein having predominantlya particular N-linked glycan structure and having reduced O-linkedglycosylation using the method disclosed herein. It has been found thatsome host cells that have been genetically modified to produceglycoproteins having predominantly a particular N-linked glycanstructure can grow less well in culture under particular conditions thanhost cells that have not been modified. For example, particular fungaland yeast cells in which genes involved in hypermannosylation have beendeleted and other genes needed to produce particular mammalian or humanlike N-linked glycan structures have been added, can grow less well thanfungal or yeast cells that do not the genetic modifications. In some ofthese genetically modified fungal or yeast cells, further introducingdeletions of the PMT genes either is lethal to the cells or adverselyaffects the ability of the cells to grow to sufficient quantities inculture. The methods herein avoid the potential deleterious effects ofdeleting the PMT genes by allowing the cells to grow to sufficientquantities in culture before inducing expression of the recombinantglycoprotein and adding an inhibitor of the activity of the Pmtproteins, optionally with one or more a 1,2-mannosidases, to produce therecombinant glycoprotein having predominantly particular N-linked glycanstructures and reduced O-linked glycosylation.

An aspect of the methods described herein is that it provides for aglycoprotein composition comprising reduced O-linked glycosylation and apredominantly a specific N-linked glycoform in which the recombinantglycoprotein may exhibit increased biological activity and/or decreasedundesired immunogenicity relative to compositions of the sameglycoprotein produced from mammalian cell culture, such as CHO cells. Anadditional advantage of producing the glycoprotein compositioncomprising reduced O-linked glycosylation and a predominant N-linkedglycoform is that it avoids production of undesired or inactiveglycoforms and heterogeneous mixtures, which may induce undesiredeffects and/or dilute the more effective glycoform. Thus, therapeuticpharmaceutical composition of glycoprotein molecules comprising, forexample, predominantly Man₅GlcNAc₂, Man₃GlcNAc₂, GlcNAcMan₅GlcNAc₂,GlcNAcMan₃GlcNAc₂, GlcNAc₂Man₃GlcNAc₂, GalGlcNAcMan₅GlcNAc₂,Gal(GlcNAc)₂ Man₅GlcNAc₂, (GalGlcNAc)₂Man₅GlcNAc₂,NANAGalGlcNAcMan₃GlcNAc₂, NANA₂Gal₂GlcNAcMan₃GlcNAc₂, andGalGlcNAcMan₃GlcNAc₂ glycoforms and having reduced O-linkedglycosylation may well be effective at lower doses, thus having higherefficacy/potency.

In general, the method for producing proteins having reduced O-linkedglycosylation comprises transforming a host cell with a nucleic acidencoding a recombinant or heterologous protein in which it is desirableto produce the protein having reduced O-linked glycosylation. Thenucleic acid encoding the recombinant protein is operably linked toregulatory sequences that allow expression of the recombinant protein.Such regulatory sequences include an inducible promoter and optionallyan enhancer upstream, or 5′, to the nucleic acid encoding the fusionprotein and a transcription termination site 3′ or down stream from thenucleic acid encoding the recombinant protein. The nucleic acid alsotypically encodes a 5′ UTR region having a ribosome binding site and a3′ untranslated region. The nucleic acid is often a component of avector replicable in cells in which the recombinant protein isexpressed. The vector can also contain a marker to allow recognition oftransformed cells. However, some cell types, particularly yeast, can besuccessfully transformed with a nucleic acid lacking extraneous vectorsequences.

Nucleic acids encoding desired recombinant proteins can be obtained fromseveral sources. cDNA sequences can be amplified from cell lines knownto express the protein using primers to conserved regions (see, forexample, Marks et al., J. Mol. Biol. 581-596 (1991)). Nucleic acids canalso be synthesized de novo based on sequences in the scientificliterature. Nucleic acids can also be synthesized by extension ofoverlapping oligonucleotides spanning a desired sequence (see, e.g.,Caldas et al., Protein Engineering, 13, 353-360 (2000)).

In one aspect, the nucleic acid encoding the protein is operably linkedto an inducible promoter, which allows expression of the protein to beinduced when desired. In another aspect, the nucleic acid encoding theprotein is operably linked to a constitutive promoter. To facilitateisolation of the expressed protein, it is currently preferable that theprotein include a signal sequence that directs the protein to beexcreted into the cell culture medium where it can then be isolated. Inthe first aspect, the transformed host cells are cultured for a timesufficient to produce a desired multiplicity of host cells sufficient toproduce the desired amount of protein before adding one or moreinhibitors of Pmt-mediated O-linked glycosylation to the culture medium.The inducer and inhibitor can be added to the culture simultaneously orthe inducer is added to the culture before adding the one or more Pmtinhibitors or the one or more Pmt inhibitors is added to the culturebefore adding the inducer. The induced protein is produced havingreduced O-linked glycosylation and can be recovered from the culturemedium or for proteins not having a signal sequence, from the host cellby lysis. In the second aspect, wherein the nucleic acid encoding theprotein is operably linked to a constitutive promoter, the one or moreinhibitors of Pmt-mediated O-linked glycosylation is added to theculture medium at the same time the culture is established and theprotein, which is produced having reduced O-linked glycosylation, can berecovered from the culture medium or for proteins not having a signalsequence, from the host cell by lysis.

Chemicals or compositions that inhibit the activity one or more of thePmt proteins useful for producing proteins with reduced O-linkedglycosylation are described herein. When the host cell is a lowereukaryote such as fungi or yeast, it is desirable that the inhibitorinhibit at least the activity of Pmt1 or Pmt2, or both. In highereukaryotes, it is desirable that the inhibitor inhibit activity of thehomologue in the higher eukaryote that corresponds to the Pmt1 or Pmt2.

The compounds of the invention are shown to be effective in producingrecombinant proteins having reduced O-linked glycosylation in Pichiapastoris strains that had intact, functional PMT genes. Table 1 and FIG.1 of Example 5 show that any one of the above four Pmt chemicalinhibitors added to a culture of recombinant Pichia pastoris havingintact, functional PMT genes and transformed with a nucleic acidencoding a recombinant, human anti-Her2 antibody protein operably linkedto an inducible promoter at the time of expression of the recombinantprotein was induced, produced a recombinant protein having a level ofreduced O-linked glycosylation that was improved relative to the levelof O-linked glycosylation seen for Pichia pastoris cells treated withPmt inhibitor Pmti-3 described in Orchard et al. (Bioorgan & Med ChemLetters (2004) 14:3975-3978) and patent publications by the same authorsincluding EP 1313471 B1 and Bobrowicz et al. U.S. Published ApplicationNo. 2007061631.

Host Cells

The term “cells” as used herein refers to host cells described asfollows. While host cells for the method herein includes both highereukaryote cells and lower eukaryote cells, lower eukaryote cells, forexample filamentous fungi or yeast cells, are currently preferred forexpression of proteins because they can be economically cultured, givehigh yields of protein, and when appropriately modified are capable ofproducing proteins having suitable glycosylation patterns. Lowereukaryotes include yeast, fungi, collar-flagellates, microsporidia,alveolates (e.g., dinoflagellates), stramenopiles (e.g, brown algae,protozoa), rhodophyta (e.g., red algae), plants (e.g., green algae,plant cells, moss) and other protists. Yeast and fungi include, but arenot limited to: Pichia sp. (for example, Pichia pastoris, Pichiafinlandica, Pichia trehalophila, Pichia koclamae, Pichiamembranaefaciens, Pichia minuta (Ogataea minuta, Pichia lindneri),Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichiaguercuum, Pichia pijperi, Pichia stiptis, Pichia methanolica),Saccharomyces sp. (for example Saccharomyces cerevisiea), Hansenulapolymorpha, Kluyveromyces sp. (for example, Kluyveromyces lactis),Candida albicans, Aspergillus sp (for example, Aspergillus nidulans,Aspergillus niger, Aspergillus oryzae), Trichoderma reesei,Chrysosporium lucknowense, Fusarium sp. (for example, Fusariumgramineum, Fusarium venenatum), Physcomitrella patens and Neurosporacrassa. Yeast, in particular, are currently preferred because yeastoffers established genetics allowing for rapid transformations, testedprotein localization strategies, and facile gene knock-out techniques.Suitable vectors have expression control sequences, such as promoters,including 3-phosphoglycerate kinase or other glycolytic enzymes, and anorigin of replication, termination sequences, and the like as desired.

Various yeasts, such as K. lactis, Pichia pastoris, Pichia methanolica,and Hansenula polymorpha are currently preferred for cell culturebecause they are able to grow to high cell densities and secrete largequantities of recombinant protein. Likewise, filamentous fungi, such asAspergillus niger, Fusarium sp, Neurospora crass, and others can be usedto produce recombinant proteins at an industrial scale.

Lower eukaryotes, in particular filamentous fungi and yeast, can begenetically modified so that they express proteins or glycoproteins inwhich the glycosylation pattern is human-like or humanized. This can beachieved by eliminating selected endogenous glycosylation enzymes and/orsupplying exogenous enzymes as described by Gerngross et al. in U.S.Pat. No. 7,029,872, and U.S. Published Patent Application Nos.20040018590, 20050170452, 20050260729, 20040230042, 20050208617,20040171826, 20050208617, 20060160179, 20060040353, and 20060211085.Thus, a host cell can additionally or alternatively be engineered toexpress one or more enzymes or enzyme activities, which enable theproduction of particular N-glycan structures at a high yield. Such anenzyme can be targeted to a host subcellular organelle in which theenzyme will have optimal activity, for example, by means of signalpeptide not normally associated with the enzyme. Host cells can also bemodified to express a sugar nucleotide transporter and/or a nucleotidediphosphatase enzyme.

The transporter and diphosphatase improve the efficiency of engineeredglycosylation steps, by providing the appropriate substrates for theglycosylation enzymes in the appropriate compartments, reducingcompetitive product inhibition, and promoting the removal of nucleosidediphosphates. See, for example, Gerngross et al. in U.S. PublishedPatent Application No. 20040018590 and Hamilton, 2003, Science 301:1244-46 and the aforementioned U.S. patent and patent applications.

By way of example, a host cell (for example, yeast or fungal) can beselected or engineered to be depleted in 1,6-mannosyl transferaseactivities, which would otherwise add mannose residues onto the N-glycanof a glycoprotein, and to further include a nucleic acid for ectopicexpression of an α-1,2 mannosidase activity, which enables production ofrecombinant glycoproteins having greater than 30 mole percentMan₅GlcNAc₂ N-glycans. When a glycoprotein is produced in the host cellsaccording to the method described herein, the host cells will produce aglycoprotein having predominantly a Man₅GlcNAc₂ N-glycan structure andreduced O-glycosylation compared to the glycoprotein produced in thecell otherwise. In a further aspect, the host cell is engineered tofurther include a nucleic acid for ectopic expression of GlcNActransferase I activity, which enables production of glycoproteins havingpredominantly GlcNAcMan₅GlcNAc₂ N-glycans. When a glycoprotein isproduced in the host cells according to the method described herein, thehost cells will produce a glycoprotein having predominantly aGlcNAcMan₅GlcNAc₂ N-glycan structure and reduced O-glycosylationcompared to the glycoprotein produced in the cell otherwise. In afurther still aspect, the host cell is engineered to further include anucleic acid for ectopic expression of mannosidase II activity, whichenables production of glycoproteins having predominantlyGlcNAcMan₃GlcNAc₂ N-glycans. When a glycoprotein is produced in the hostcells according to the method described herein, the host cells willproduce a glycoprotein having predominantly a GlcNAcMan₃GlcNAc₂ N-glycanstructure and reduced O-glycosylation compared to the glycoproteinproduced in the cell otherwise. In a further still aspect, the host cellis engineered to further include a nucleic acid for ectopic expressionof GlcNAc transferase II activity, which enables production ofglycoproteins having predominantly GlcNAc₂Man₃GlcNAc₂ N-glycans. When aglycoprotein is produced in the host cells according to the methoddescribed herein, the host cells will produce a glycoprotein havingpredominantly a GlcNAc₂Man₃GlcNAc₂ N-glycan structure and reducedO-glycosylation compared to the glycoprotein produced in the cellotherwise. In further still aspects, the above host cells can be furtherengineered to produce particular hybrid or complex N-glycan orhuman-like N-glycan structures by further including one or more highereukaryote genes involved in N-linked glycosylation, in any combination,that encode for example, sialytransferase activities, class II and IIImannosidase activities, GlcNAc transferase II, III, IV, V, VI, IXactivity, and galactose transferase activity. It is currently preferablethat the cells further include one or more of nucleic acids encodingUDP-specific diphosphatase activity, GDP-specific diphosphataseactivity, and UDP-GlcNAc transporter activity.

Plants and plant cell cultures may be used for expression of proteinsand glycoproteins with reduced O-linked glycosylation as taught herein(See, for example, Larrick & Fry, 1991, Hum. Antibodies Hybridomas 2:172-89); Benvenuto et al., 1991, Plant Mol. Biol. 17: 865-74); Durin etal., 1990, Plant Mol. Biol. 15: 281-93); Hiatt et al., 1989, Nature 342:76-8). Preferable plant hosts include, for example, Arabidopsis,Nicotiana tabacum, Nicotiana rustica, and Solanum tuberosum.

Insect cell culture can also be used to produce proteins andglycoproteins proteins and glycoproteins with reduced O-linkedglycosylation, as taught herein for example, baculovirus-basedexpression systems (See, for example, Putlitz et al., 1990,Bio/Technology 8: 651-654).

Although not currently as economical to culture as lower eukaryotes andprokaryotes, mammalian tissue cell culture can also be used to expressand produce proteins and glycoproteins with reduced O-linkedglycosylation as taught herein (See Winnacker, From Genes to Clones (VCHPublishers, NY, 1987). Suitable hosts include CHO cell lines, variousCOS cell lines, HeLa cells, preferably myeloma cell lines or the like ortransformed B-cells or hybridomas. Expression vectors for these cellscan include expression control sequences, such as an origin ofreplication, a promoter, an enhancer (Queen et al., 19861, mmunol. Rev.89:49-68), and necessary processing information sites, such as ribosomebinding sites, RNA splice sites, polyadenylation sites, andtranscriptional terminator sequences. Expression control sequences arepromoters derived from immunoglobulin genes, SV40, Adenovirus, bovinePapilloma Virus, cytomegalovirus and the like. Generally, a selectablemarker, such as a neoR expression cassette, is included in theexpression vector.

The nucleic acid encoding the protein to be expressed can be transferredinto the host cell by conventional methods, which vary depending on thetype of cellular host. For example, calcium phosphate treatment,protoplast fusion, natural breeding, lipofection, biolistics,viral-based transduction, or electroporation can be used for cellularhosts. Tungsten particle ballistic transgenesis is preferred for plantcells and tissues. (See, generally, Maniatis et al., Molecular Cloning:A Laboratory Manual (Cold Spring Harbor Press, 1982))

Once expressed, the proteins or glycoproteins having reduced O-linkedglycosylation can be purified according to standard procedures of theart, including ammonium sulfate precipitation, affinity columns, columnchromatography, gel electrophoresis and the like (See, generally,Scopes, R., Protein Purification (Springer-Verlag, N.Y., 1982)).Substantially pure glycoproteins of at least about 90 to 95% homogeneityare preferred, and 98 to 99% or more homogeneity most preferred, forpharmaceutical uses. Once purified, partially or to homogeneity asdesired, the proteins can then be used therapeutically (includingextracorporeally) or in developing and performing assay procedures,immunofluorescent stainings, and the like. (See, generally,Immunological Methods, Vols. I and II (Lefkovits and Pernis, eds.,Academic Press, NY, 1979 and 1981).

Therefore, further provided are glycoprotein compositions comprising apredominant species of N-glycan structure and having reduced O-linkedglycosylation compared to compositions of the glycoprotein which havebeen produced in host cells have not been incubated in the presence ofan inhibitor of Pmt-mediated O-linked glycosylation or anα-1,2-mannosidase capable of trimming more than one mannose residue froma glycans structure or both. In particular aspects, the glycoproteincomposition comprises a glycoprotein having a predominant N-glycanstructure selected from the group consisting of Man₅GlcNAc₂,Man₃GlcNAc₂, GlcNAcMan₅GlcNAc₂, GlcNAcMan₃GlcNAc₂, GlcNAc₂Man₃GlcNAc₂,GalGlcNAcMan₅GlcNAc₂, Gal(GlcNAc)₂ Man₅GlcNAc₂, (GalGlcNAc)₂Man₅GlcNAc₂,NANAGalGlcNAcMan₃GlcNAc₂, NANA₂Gal₂GlcNAcMan₃GlcNAc₂, andGalGlcNAcMan₃GlcNAc₂ glycoforms.

Pharmaceutical Compositions

Proteins and glycoproteins having reduced O-linked glycosylation can beincorporated into pharmaceutical compositions comprising theglycoprotein as an active therapeutic agent and a variety of otherpharmaceutically acceptable components (See, Remington's PharmaceuticalScience (15th ed., Mack Publishing Company, Easton, Pa., 1980). Thepreferred form depends on the intended mode of administration andtherapeutic application. The compositions can also include, depending onthe formulation desired, pharmaceutically-acceptable, non-toxic carriersor diluents, which are defined as vehicles commonly used to formulatepharmaceutical compositions for animal or human administration. Thediluent is selected so as not to affect the biological activity of thecombination. Examples of such diluents are distilled water,physiological phosphate-buffered saline, Ringer's solutions, dextrosesolution, and Hank's solution. In addition, the pharmaceuticalcomposition or formulation can also include other carriers, adjuvants,or nontoxic, nontherapeutic, nonimmunogenic stabilizers, and the like.

Pharmaceutical compositions for parenteral administration are sterile,substantially isotonic, pyrogen-free and prepared in accordance with GMPof the FDA or similar body. Glycoproteins can be administered asinjectable dosages of a solution or suspension of the substance in aphysiologically acceptable diluent with a pharmaceutical carrier thatcan be a sterile liquid such as water oils, saline, glycerol, orethanol. Additionally, auxiliary substances, such as wetting oremulsifying agents, surfactants, pH buffering substances and the likecan be present in compositions. Other components of pharmaceuticalcompositions are those of petroleum, animal, vegetable, or syntheticorigin, for example, peanut oil, soybean oil, and mineral oil. Ingeneral, glycols such as propylene glycol or polyethylene glycol arepreferred liquid carriers, particularly for injectable solutions.Glycoproteins can be administered in the form of a depot injection orimplant preparation which can be formulated in such a manner as permit asustained release of the active ingredient. Typically, compositions areprepared as injectables, either as liquid solutions or suspensions;solid forms suitable for solution in, or suspension in, liquid vehiclesprior to injection can also be prepared. The preparation also can beemulsified or encapsulated in liposomes or micro particles such aspolylactide, polyglycolide, or copolymer for enhanced adjuvant effect,as discussed above (See Langer, Science 249, 1527 (1990) and Hanes,Advanced Drug Delivery Reviews 28, 97-119 (1997).

The term “or a salt thereof” refers to salts prepared from acceptablebases including inorganic or organic bases. Salts derived from inorganicbases include aluminum, ammonium, calcium, copper, ferric, ferrous,lithium, magnesium, manganic salts, manganous, potassium, sodium, zinc,and the like. Salts in the solid form may exist in more than one crystalstructure, and may also be in the form of hydrates. Salts derived frombases include salts of primary, secondary, and tertiary amines,substituted amines including naturally occurring substituted amines,cyclic amines, and basic ion exchange resins, such as arginine, betaine,caffeine, choline, N,N′-dibenzylethylene-diamine, diethylamine,2-diethylaminoethanol, 2-dimethylaminoethanol, ethanolamine,ethylenediamine, N-ethyl-morpholine, N-ethylpiperidine, glucamine,glucosamine, histidine, hydrabamine, isopropylamine, lysine,methylglucamine, morpholine, piperazine, piperidine, polyamine resins,procaine, purines, theobromine, triethylamine, trimethylamine,tripropylamine, tromethamine, and the like.

The compounds of the present invention may contain one or moreasymmetric centers and can thus occur as racemates and racemic mixtures,single enantiomers, diastereomeric mixtures and individualdiastereomers. The present invention is meant to comprehend all suchisomeric forms of these compounds.

Unless otherwise defined herein, scientific and technical terms andphrases used in connection with the present invention shall have themeanings that are commonly understood by those of ordinary skill in theart. Further, unless otherwise required by context, singular terms shallinclude the plural and plural terms shall include the singular.Generally, nomenclatures used in connection with, and techniques ofbiochemistry, enzymology, molecular and cellular biology, microbiology,genetics and protein and nucleic acid chemistry and hybridizationdescribed herein are those well known and commonly used in the art. Themethods and techniques of the present invention are generally performedaccording to conventional methods well known in the art and as describedin various general and more specific references that are cited anddiscussed throughout the present specification unless otherwiseindicated. See, for example, Sambrook et al. Molecular Cloning: ALaboratory Manual, 2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989); Ausubel et al., Current Protocols inMolecular Biology, Greene Publishing Associates (1992, and Supplementsto 2002); Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory Press, Cold Spring Harbor, N.Y. (1990); Taylor andDrickamer, Introduction to Glycobiology, Oxford Univ. Press (2003);Worthington Enzyme Manual, Worthington Biochemical Corp., Freehold,N.J.; Handbook of Biochemistry: Section A Proteins, Vol I, CRC Press(1976); Handbook of Biochemistry: Section A Proteins, Vol II, CRC Press(1976); Essentials of Glycobiology, Cold Spring Harbor Laboratory Press(1999).

The following examples provide methods for preparing various Pmtinhibitors of the invention.

Example 1

Step A

To a solution of 1-1 (5.90 g, 25 mmol) in THF (200 mL) was added NaH(2.0 g, 50 mmole) portionwise. The resulting mixture was stirred at 0°C. for half an hour. Then NBS (4.86 g, 27.5 mmol) was added into andstirred for 15 minutes. The white solid was filtered and the filtrationwas concentrated to give a residue, which was dissolved in CHCl₃ anddried over Na₂SO₄. The solvent was evaporated to give 1-2 (5 g, yield63%), which was used for next step without further purification.

Step B

To a solution of 1-2 (1.8 g, 6 mmol) and 1-3 (1.3 g, 5 mmol) in driedDMF (5 mL) was added NaH (2.4 g g, 10 mmol) and then stirred at roomtemperature overnight. The resulting mixture was partitioned betweenwater and EtOA. The combined organic layer were washed with brine, driedover sodium sulfate and concentrated. The residue was purified byprep-TLC to give 1-4 (800 mg, yield 40%). ¹H-NMR (400 MHz, CDCl₃) δ 9.66(s, 1H), 7.74˜7.71 (m, 2H), 7.49˜1.46 (m, 1H), 7.37˜7.33 (m, 5H), 7.20(d, J=2.02 Hz, 1H), 6.97˜6.92 (m, 3H), 4.28˜4.19 (m, 6H), 3.16 (t, J=6.6Hz, 2H), 1.11 (m, 6H).

Step C

To a suspension of LAH (380 mg, 10 mmol) in THF (50 mL) was added 1-4(1.3 g, 2.6 mmol) at 0° C. and the resulting mixture was stirred at roomtemperature for 30 min. The mixture was carefully treated by diluted aq.HCl and then partitioned between water and EtOAc. The organic layer waswashed by brine, dried over sodium sulfate and concentrated. The residuewas purified by prep-TLC to give 1-5 (260 mg, 20% yield).

Step D

To a solution of 1-5 (300 mg, 0.54 mmol) in 2,2-dimethoxylpropane (1 mL)was added catalytic amount TsOH.H₂O (˜10 mg) and then the resultingmixture was stirred for one hour at room temperature. The mixture waspurified directly by prep-TLC to give 1-6 (100 mg, yield 28%). ¹H-NMR(400 MHz, MeOD) δ ppm 7.52 (d, J=8.05 Hz, 2H), 7.40˜7.15 (m, 5H),6.90˜6.85 (m, 2H), 6.80˜6.75 (m, 2H), 4.25 (s, 2H), 4.23 (m, 6H), 3.0(m, 2H), 1.48 (t, 6H).

Step E

To a solution of 1-6 (100 mg, 0.22 mmol) in dried DCM (25 mL) was addedMnO₂ (174 mg, 2 mmol) in one portion and the resulting mixture washeated to reflux for about 2 hrs. After cooling to room temperature, themixture was filtered and the filtration was concentrated and the residuewas purified by prep-TLC to give 1-7 (60 mg, yield 60%). ¹H-NMR (400MHz, MeOD) δ ppm 9.5 (s, 1H), 7.52 (d, J=7.2 Hz, 1H), 7.48 (d, J=2.8 Hz,2H), 7.30 (m, 3H), 7.25 (m, 6H), 7.0 (t, J=8.6 Hz, 2H), 6.89 (m, J=8.6Hz, 2H), 6.76 (d, J=8 Hz, 1H), 4.23 (m, 6H), 3.0 (m, 2H), 1.48 (t, 6H).

Step F

To a solution of 1-7 (80 mg, 0.17 mmol) in THF was added 6 N HCl and theresulting mixture was stirred at room temperature for 2 hours. Thesolvent was removed in vacuo to afford the crude 1-8 (60 mg, 82% yield),which was subjected to next step without further purification.

Step G

To a solution of 1-8 (60 mg, 0.146 mmol) in DMF (6 mL) was added NaH (24mg, 1 mmol) at ° C. and the resulting mixture was stirred at the sametemperature for about 30 min and then CH₃I (0.2 mL) was added into. Themixture was stirred at the same temperature for another one hour. Themixture was then partitioned between H₂O and EtOAc. The combined organiclayers were washed with brine, dried over sodium sulfate, andconcentrated. The residue was purified by prep-TLC to give 1-9 (40 mg,yield 63.0%). ¹H-NMR (400 MHz, MeOD) S ppm 9.6 (s, 1H), 7.52 (d, J=8.08Hz, 2H), 7.43 (m, 1H), 7.36-7.30 (m, 5H), 7.16 (d, J=2.02 Hz, 1H), 7.0(t, J=8.6 Hz, 2H), 6.89 (m, J=8.6 Hz, 2H), 4.23 (t, J=6.56 Hz, 2H),3.95-3.85 (m, 4H), 3.30 (s, 6H), 3.13 (dd, J₁=6.26 Hz, J₂=6.26 Hz, 2H).

Step H

A mixture of 1-9 (40 mg, 0.09 mmol), 1-10 (19 mg, 0.09 mmol) and NH₄OAc(70 mg, 0.9 mmol) in toluene (10 mL) was heated under nitrogen to refluxfor about 3 hrs. After cooling to room temperature, the resultingmixture was treated by diluted aq. HCl until pH=4˜5 and then partitionedbetween H₂O and EtOAc. The combined organic layers were washed by brine,dried over Na₂SO₄ and concentrated. The residue was purified byprep-HPLC to give EXAMPLE 1 (30 mg, yield 50%) as a yellow solid. ¹H-NMR(400 MHz, MeOD) δ ppm 7.46˜7.53 (m, 3H), 7.31˜7.40 (m, 5H), 7.15˜7.18(m, 1H), 6.92˜7.06 (m, 4H), 4.71 (s, 2H), 4.25˜4.30 (m, 2H), 3.85˜3.94(m, 4H), 3.26˜3.29 (m, 6H), 3.10˜3.13 (dd, J=6.26 Hz, J=6.26 Hz, 2H). MSm/z 612(M+1)⁺.

Example 2

Step A

To a solution of compound 2-1 (150 mg, 0.58 mmol, 1.0 eq), compound 2-2(214 mg, 0.87 mmol, 1.5 eq) in DMF (10 mL) was added Cs₂CO₃ (161 mg,0.49 mmol, 0.85 eq). The mixture was then stirred at room temperatureovernight and then heated to 80° C. for 1.5 hrs. The resulting mixturewas then partitioned between H₂O and EtOAc. The combined organic layerswere washed with brine and dried over Na₂SO₄, concentrated and purifiedby prep-TLC to give 2-3 (122 mg, yield 49.4%).

Step B

A mixture of compound 2-3 (122 mg, 0.29 mmol, 1.0 eq), 2-4 (57 mg, 0.30mmol, 1.05 eq) and NH₄OAc (223 mg, 2.90 mmol, 10.0 eq) in toluene (15mL) was heated under nitrogen to reflux for about 3 h. The resultingmixture was treated by 10% HCl acid to pH=4-5, and then partitionedbetween H₂O and EtOAc. The combined organic layers were washed withbrine and dried over Na₂SO₄, concentrated and purified by prep-TLC togive EXAMPLE 2 (52 mg, yield 29.9%). ¹H-NMR (400 MHz, MeOD) δ 7.41-7.47(m, 3H), 7.19-7.35 (m, 10H), 6.99-7.07 (m, 3H), 6.87-6.92 (m, 2H), 6.22(s, 1H), 4.75 (s, 2H), 4.24-4.29 (m, 2H), 3.72-3.13 (m, 2H). MS m/z 600(M+1)⁺

Example 3

Step A

To a solution of 3-1 (1 g, 6.37 mmol) in dry THF (20 mL) was added BuLi(3 mL, 7.68 mmol) dropwise at −70° C. and then the mixture was stirredfor 30 min at room temperature. After cooling to −70° C. again, asolution of cyclohexanecarbaldehyde (0.72 g, 6.43 mmol) in dry THF (5mL) was added dropwise. After stirring for 30 min, saturated aqueousNH₄Cl was added into the mixture and the resulting mixture was extractedwith EtOAc. The organic layers were combined and dried over Na₂SO₄,concentrated to give 3-2 (1.52 g, 14.3%) as a crude oil.

Step B

To the mixture of 3-2 (600 mg, 3.1 mmol) in dry DCM (15 mL) was addedSOCK (2 mL) dropwise at 0° C. Then the resulting mixture was stirred for2 h at room temperature. The mixture was concentrated under reducedpressure and then diluted with EtOAc. The organic layer was washed withsaturated aqueous NaHCO₃, brine, dried over Na₂SO₄, and thenconcentrated to give 3-3 (500 mg, crude).

Step C

To a solution of 3-3 (450 mg, crude) and 3-4 (375 mg, 1.44 mmol) in DMF(10 mL) was added Cs₂CO₃ (399 mg, 1.22 mmol) in one portion and then themixture was heated to 110° C. for 18 h. The mixture was diluted with H₂Oand extracted with EtOAc. The organic layers were combined and driedover Na₂SO₄, concentrated. The residue was purified by TLC to afford 3-5(130 mg) as oil, which was used for next step directly.

Step D

To a mixture of 3-5 (130 mg, 0.3 mmol) and 3-6 (60 mg, 0.31 mmol) intoluene (15 mL) was added NH₄OAc (180 mg, 2.34 mmol) in one portion andthen the mixture was heated to reflux for 3.5 h. Then the mixture wascooled to room temperature and aqueous 10% HCl was added to pH=5. Themixture was extracted with EtOAc. The organic layers were combined anddried over Na₂SO₄, concentrated to give yellow oil. The residue waspurified by prep-TLC to afford EXAMPLE 3 (150 mg, yield 82.4%). ¹H-NMR(300 MHz, MeOD) δ 7.60 (s, 1H), 7.19-7.49 (m, 11H), 6.88 (s, 1H), 6.81(s, 1H), 5.05 (s, 1H), 4.65 (s, 2H), 4.25˜4.39 (m, 2H), 3.15 (m, 2H),1.60˜4.98 (m, 5H), 1.00˜4.46 (m, 6H). MS m/z: 607 (M+1)⁺.

Example 4

Step A

Sodium (8.5 mg, 0.37 mmol) was dissolved at 40° C. in2-methyl-propan-1-ol (1.3 g, 17.5 mmol) and compound 4-1 (2 g, 16.7mmol) were subsequently added dropwise at 50° C. over 30 min. Then thereaction mixture was heated to 70° C. and stirred overnight. Theresulting mixture was partitioned between H₂O and EtOAc. The organiclayer was washed with brine, dried over sodium sulfate and concentratedto give 4-2 (1.0 g, crude), which is used for the next step directly.

Step B

To a solution of 4-2(1.0 g, crude), 4-3 (400 mg, 1.64 mmol, 1.0 eq) andPPh₃ (516 mg, 1.96 mmol, 1.2 eq) in THF (20 mL) was added DIAD (406 mg,1.96 mmol, 1.2 eq) dropwise at 0° C. The resulting mixture was stirredat room temperature overnight and then partitioned between H₂O andEtOAc. The organic layer was washed by brine, dried over Na₂SO₄,concentrated and purified by prep-HPLC to give 4 (90 mg, yield 12.0%).¹H-NMR (400 MHz, MeOD) δ 9.72 (s, 1H), 7.28-7.40 (m, 9H), 6.98-7.05 (t,J=8.78 Hz, 2H), 6.89-6.92 (d, J=8.28 Hz, 1H), 5.42-5.46 (m, 1H),4.22-4.32 (m, 2H), 3.86-3.92 (m, 1H), 3.68-3.74 (m, 1H), 3.24-3.34 (m,2H), 3.12-3.18 (m, 2H), 1.82-1.90 (m, 1H), 0.84-0.88 (m, 6H).

Step C

The preparation of EXAMPLE 4 is the same as that for EXAMPLE 3. ¹H-NMR(400 MHz, MeOD) δ 7.49 (s, 1H), 7.24-7.40 (m, 7H), 6.98-7.05 (m, 4H),6.90-6.91 (m, 1H), 5.37-5.40 (m, 1H), 4.75 (s, 2H), 4.21-4.32 (m, 2H),3.83-3.88 (m, 1H), 3.69-3.72 (m, 1H), 3.33-3.35 (m, 2H), 3.09-3.13 (t,J=6.57 Hz, 2H), 1.79-1.87 (m, 1H), 0.86-0.88 (m, 6H).

Example 5

This example shows that Pichia pastoris transformed with an expressionvector encoding the heavy chain (Hc) and light chain (Lc) of the humananti-Her2 antibody and treated with the novel Pmt inhibitors describedherein produced a glycoprotein having reduced O-glycosylation.

Expression/integration plasmid vector pGLY2988 contains expressioncassettes under control of the methanol-inducible Pichia pastoris AOX1promoter that encode the heavy (Hc) and light (Lc) chains of anti-Her2.Anti-Her2 Hc and Lc fused at the N-terminus to α-MAT pre signal peptide(SEQ ID Nos:1 and 2) were synthesized by GeneArt AG. Each wassynthesized with unique 5′ EcoR1 and 3′ Fse1 sites. The nucleotide andamino acid sequences of the anti-Her2 Hc are shown in SEQ ID Nos:3 and4, respectively. The nucleotide and amino acid sequences of theanti-Her2 Lc are shown in SEQ ID Nos:5 and 6, respectively. Both nucleicacid fragments encoding the Hc and Lc proteins fused to the a-MAT presignal peptide were separately subcloned using 5′ EcoR1 and 3′ Fse1unique sites into an expression plasmid vector pGLY2198, which containsthe Pichia pastoris TRP2 targeting nucleic acid and theZeocin-resistance marker and generates expression cassettes under thecontrol of the AOX1 promoter and Saccharomyces cerevisiae CYCterminator, to form plasmid vectors pGLY2987 and pGLY2338, respectively.The Lc expression cassette was then removed from plasmid vector pGLY2338by digesting with BamHI and NotI and subcloned into plasmid vectorpGLY2987 digested with BamH1 and Not1, thus generating the finalexpression plasmid vector pGLY2988.

Anti-Her2 expression strain yGLY4280 was constructed as follows: Fivemicrograms of pGLY2988 digested with restriction enzyme Spe1 which cutsin the TRP2 targeting region were used to transform strain yGLY22-1.Strain yGLY22-1 (och1Δ::lacZbmt2Δ::lacZ/K1MNN2-2/mnn4L1Δ::lacZ|MmSLC35A3 pnol Δmnn4Δ::lacZmet16Δ::lacZ), was constructed using methods described earlier (Nett andGemgross, Yeast 20:1279 (2003); Choi et al., PNAS USA 100:5022 (2003);Hamilton et al., Science 301:1244 (2003)).

Transformation of yGLY22-1 performed essentially as follows: YGLY22-1was grown in 50 mL YPD media (yeast extract (1%), peptone (2%), dextrose(2%)) overnight to an OD of between about 0.2 to 6; After incubation onice for 30 minutes, cells were pelleted by centrifugation at 2500-3000rpm for 5 minutes. Media was removed and the cells washed three timeswith ice cold sterile 1M sorbitol before resuspension in 0.5 ml ice coldsterile 1M sorbitol. Ten μL of linearized DNA (10 ug) and 100 mL cellsuspension was combined in an electroporation cuvette and incubated for5 minutes on ice. Electroporation was in a Bio-Rad GenePulser Xcellfollowing the preset Pichia pastoris protocol (2 kV, 25 μF, 200Ω),immediately followed by the addition of 1 mL YPDS recovery media (YPDmedia plus 1 M sorbitol). The transformed cells were allowed to recoverfor four hours to overnight at room temperature (26° C.) before platingthe cells on the selective media. Following selection on mediacontaining zeocin, transformants were screened by small scale expressionanalysis to detect anti-Her2 expression. Strain yGLY4280 was selectedbased on high level anti-Her2 expression.

Anti-Her2 protein expression for strain yGLY4280 was carried out inshake flasks at 24° C. with buffered glycerol-complex medium (BMGY)consisting of 1% yeast extract, 2% peptone, 100 mM potassium phosphatebuffer pH 6.0, 1.34% yeast nitrogen base, 4×10-5% biotin, and 1%glycerol. The induction medium for protein expression was bufferedmethanol-complex medium (BMMY) consisting of 1% methanol instead ofglycerol in BMGY. Pmt inhibitors in 100% methanol were added to thegrowth medium to a final concentration of 0.15 ug/mL at the time theinduction medium was added. This is an intermediate dose that issufficient to reduce O-glycosylation to roughly 50% of that observedwithout Pmt inhibitor treatment, and thus allows a comparison of potencyfor the different inhibitors. Following 24 hr further growth in theinduction media, cultures were harvested and centrifuged at 2,000 rpmfor five minutes to remove cells from the supernatant.

O-glycan determination was performed using a Dionex-HPLC(HPAEC-PAD) asfollows. To measure O-glycosylation reduction, protein was purified fromthe growth medium using protein A chromatography (Li et al. Nat.Biotechnol. 24(2):210-5 (2006)) and the O-glycans released from andseparated from protein by alkaline elimination (beta-elimination)(Harvey, Mass Spectrometry Reviews 18: 349-451 (1999)). This processalso reduces the newly formed reducing terminus of the released O-glycan(either oligomannose or mannose) to mannitol. The mannitol group thusserves as a unique indicator of each O— glycan. 0.5 nmole or more ofprotein, contained within a volume of 100 μL PBS buffer, was requiredfor beta elimination. The sample was treated with 25 μL alkalineborohydride reagent and incubated at 50° C. for 16 hours. About 20 uLarabitol internal standard was added, followed by 10 μL glacial aceticacid. The sample was then centrifuged through a Millipore filtercontaining both SEPABEADS and AG 50W-X8 resin and washed with water. Thesamples, including wash, were transferred to plastic autosampler vialsand evaporated to dryness in a centrifugal evaporator. 150 μL 1%AcOH/MeOH was added to the samples and the samples evaporated to drynessin a centrifugal evaporator. This last step was repeated five moretimes. 200 μL of water was added and 100 μL of the sample was analyzedby high pH anion-exchange chromatography coupled with pulsedelectrochemical detection-Dionex HPLC(HPAEC-PAD). Average O-glycanoccupancy was determined based upon the amount of mannitol recovered.

The results are summarized in Table 1, which shows that the four novelPmt inhibitors, Examples 1 to 4, reduce O-glycosylation to levels lowerthat that observed with Pmt inhibitor Pmti-3 as described in Orchard etal. (Bioorgan & Med Chem Letters (2004) 14:3975-3978) and patentpublications by the same authors, including EP 1313471 B1 and Bobrowiczet al., U.S. Published Application No. 2007061631. Pmti-3 has thefollowing chemical structure:

TABLE 1 Inhibitor O-glycan occupancy (0.15 ug/mL) (moles O-mannose/molesAb) none >14.0 Pmti-3 8.1 Example 1 4.1 Example 2 4.3 Example 3 5.5Example 4 5.2

Visual proof that the novel Pmt inhibitors effectively reduceO-glycosylation of Pichia-produced recombinant protein is provided byFIG. 1, which shows the effects of increasing amounts of Pmt inhibitorson O-glycosylation of anti-Her2 heavy chain (Hc). Strain yGLY4280 wasinoculated into 96-well deep well plates (Qiagen, Valencia, Calif.)containing 0.5 ml of BMGY media per well. After 24 hours growth withvigorous shaking, the 96-well plate was centrifuged at 2,000 rpm forfive minutes to pellet cells. The media was removed and, following awash step with 0.5 mL of BMMY media, the cells resuspended in 0.2 mLBMMY media in which Pmt inhibitors were diluted 2-fold across the rows(11 wells). Well #1 contained 5 ug/mL of inhibitor, well #2 contained2.5 ug/mL and so on until well #10 contained 0.009 ug/mL; well #11contained no inhibitor. After an additional 24 hours growth withvigorous shaking, the plate was centrifuged at 2,000 rpm for fiveminutes to pellet cells, and the cleared supernatant subjected toWestern blot analysis to detect anti-Her2 expression. The Westernblotting was performed as follows: seven μL of the supernatants wereseparated by reducing polyacrylamide gel electrophoresis (SDS-PAGE)according to Laemmli, U. K. (1970) Nature 227, 680-685 and thenelectroblotted onto nitrocellulose membranes (Schleicher & Schuell, nowWhatman, Inc., Florham Park, N.J.). Anti-Her2 antibody chains weredetected on the Western blots using a peroxidase-conjugated anti-humanHc and Lc antibody (Calbiochem/EMD Biosciences, La Jolla, Calif.) anddeveloped using the ImmunoPure Metal Enhanced DAB Substrate Kit (PierceBiotechnology, Rockford, Ill.). FIG. 1 shows the results of one suchanalysis in which the novel Pmt inhibitor from Example 4 (EX. 4, panelA) was tested against. Pmti-3 from Orchard et al. (panel B) and also aninactive compound as a control (panel C). As shown in panels A and B,Example 4 and Pmti-3 effectively reduced O-glycosylation of anti-Her2 Hcat concentrations as low as 0.018 ug/mL. In contrast, the inactivecontrol compound (panel C) showed no reduction in Hc O-glycosylation.Similar results were obtained with inhibitors shown in Examples 1, 2,and 3. Taken together, these results indicate that the novel Pmtinhibitors shown in Examples 1 to 4 are effective inhibitors of fungalO-glycosylation.

While the present invention is described herein with reference toillustrated embodiments, it should be understood that the invention isnot limited hereto. Those having ordinary skill in the art and access tothe teachings herein will recognize additional modifications andembodiments within the scope thereof. Therefore, the present inventionis limited only by the claims attached herein.

1. A compound represented by the following structural formula:

or a salt thereof.
 2. A method of producing a protein having reducedO-linked glycosylation comprising: (a) growing a cell in a culture thatproduces the protein; (b) contacting the culture with one or morecompounds of claim 1, which inhibit Pmt-mediated O-linked glycosylation;and (c) isolating the protein produced by the host cell.
 3. The methodof claim 2 wherein the culture of the cell is provided by: (a) providinga nucleic acid encoding a protein; and (b) introducing the nucleic acidinto the cell.
 4. The method of claim 3 wherein the culture is grown fora time sufficient to provide a multiplicity of the cells having thenucleic acid before contacting the culture with any of the one or morecompounds which inhibit Pmt-mediated O-linked glycosylation.
 5. Themethod of claim 3 wherein the culture is grown in the presence of any ofthe one or more compounds which inhibit Pmt-mediated O-linkedglycosylation.
 6. The method of claim 3 wherein the nucleic acid isoperably linked to an inducible promoter.
 7. The method of claim 6wherein the culture is grown for a time sufficient to provide amultiplicity of the cells having the nucleic acid before contacting theculture with the one or more compounds which inhibit Pmt-mediatedO-linked glycosylation and an inducer of the promoter to induceexpression of the protein and isolating the protein produced by the cellin the presence of the one or more inhibitors and the inducer to producethe protein having reduced O-linked glycosylation.
 8. The method ofclaim 6 wherein the culture is contacted with an inducer of the promoterto induce expression of the protein for a time before contacting theculture with the one or more compounds which inhibit Pmt-mediatedO-linked glycosylation and isolating the protein produced by the cell inthe presence of the one or more compounds which inhibit Pmt-mediatedO-linked glycosylation and the inducer to produce the protein havingreduced O-linked glycosylation.
 9. The method of claim 3, wherein thecell is a fungal cell.
 10. The method of claim 3, wherein the cell is ayeast cell.
 11. The method of claim 3, wherein the cell is selected fromthe group consisting of K. lactis, Pichia pastoris, Pichia methanolica,and Hansenula.
 12. The method of claim 3, wherein the cell is Pichiapastoris.
 13. The method of claim 3, wherein the cell is a yeast orfilamentous fungal cell that has been genetically modified to produceglycoproteins with a predominant N-glycan glycoform.
 14. The method ofclaim 3 wherein the cells have been genetically modified to produceglycoproteins in which the N-glycosylation pattern is human-like orhumanized.
 15. The method of claim 3 wherein the protein is produced ata yield of at least 100 mg/liter of culture medium.